Our long-term objective is to use the advantages of the zebrafish model system to investigate how the vertebrate endoderm is regionalized such that endocrine and digestive organs, including the pancreas, are specified and develop in the correct location. The results of our studies may ultimately facilitate experiments to direct human stem cells to differentiate into specific pancreatic cell types in vitro. Such approaches offer promise for transplantation therapies for diabetes and pancreatic cancers. Our general strategy is to test the hypothesis that molecular mechanisms known to be important in regionalization of the neural ectoderm are also important for endoderm regionalization. In support of this hypothesis, we have established that the secreted signaling molecule retinoic acid (RA), which plays an important role in regionalization of both neural ectoderm and mesoderm, is critical to endoderm regionalization. Thus, RA is required for specification of the pancreas, and exogenous RA has the remarkable capacity to cause anterior endoderm to form large numbers of ectopic pancreatic cells. We propose specific aims to: (1) Determine whether RA is required for pancreas specification in tetrapod vertebrates; (2) Investigate the molecular and cellular mechanism of RA signal transduction during zebrafish pancreas specification; (3) Test the hypothesis that Hox and Parahox genes regionalize the endoderm; and (4) Investigate the roles of Wnt and FGF signaling molecules in regionalization of the endoderm. RA signals may be received in the endoderm where they act cell-autonomously to specify pancreas, or alternatively may be received in adjacent tissues, which then send secondary signals to the endoderm. We will use a cell transplantation approach to distinguish between these hypotheses. Hox genes, and related Parahox genes, encode evolutionarily conserved transcription factors. The vertebrate Hox genes regionalize ectoderm and mesoderm and are frequently RA targets. We are in the unique position of having cDNAs to all the zebrafish Hox genes, this will allow us to use gain and loss-of-function approaches (Mrna mis-expression and morpholino knockdown) to determine whether Hox genes also act to regionalize the endoderm. Finally, we will use a combination of knockdown, dominant-negative, and mis-expression approaches to investigate whether other secreted signaling molecules implicated in posteriorizing the neural ectoderm, FGFs and Wnts, are also involved in endoderm regionalization.